Gene expression changes in embryonic PECAM-1+ cells from Gata2 +9.5 enhancer targeted mice

We have generated a nonconditionall deletion of an E-box - GATA motif composite element from a putative enhancer 9.5 kb downstream of the Gata2 1S promoter. The mutation results in lethality at E13.5 to E14.5 with embryos exhibiting dectects in definitive hematopoiesis and vasculature integrity. Hematopoietic stem and progenitor cells are nearly absent from these mice and Gata2 expression is severely reduced in Pecam-1+ endothelial cells from E12.5-12.5 embryos but is expressed at normal levels in the embryonic brain. We have employed gene expression profiling in Pecam-1+ cells from wild type and Gata 2+9.5 mutant littermates to identify genes involved in maintaining vascular integrity. The Gata2+9.5 mutation was backcrossed into the C57Bl/6J background. Anti-CD31 antibody bound to magnetic beads was used to enrich for PECAM-1+ cells from four Gata2+9.5 E12.5 embryos and four wild type littermates from two litters. A single RNA sample from each embryo was used for array hybridization.

我们从Gata2 1S启动子下游9.5 kb处的推定增强子中，构建了E-box-GATA基序复合元件（E-box-GATA motif composite element）的非条件性敲除模型。该突变会导致胚胎在胚胎发育第13.5天至14.5天死亡，突变胚胎表现出定型造血功能异常与血管完整性缺陷。此类突变小鼠体内几乎不存在造血干细胞与祖细胞，且在胚胎发育第12.5天的PECAM-1（Pecam-1）阳性内皮细胞中，Gata2的表达水平显著降低，但在胚胎脑组织中其表达仍维持正常水平。为鉴定参与维持血管完整性的相关基因，我们对野生型与Gata2+9.5突变型同窝仔鼠的PECAM-1阳性细胞开展了基因表达谱分析。本研究中将Gata2+9.5突变位点回交至C57BL/6J遗传背景。实验采用磁珠偶联抗CD31抗体，从两窝共8只胚胎发育第12.5天的幼崽（4只Gata2+9.5突变型、4只野生型同窝仔鼠）中富集PECAM-1阳性细胞。每只胚胎的RNA样本单独用于芯片杂交实验。